1. Field of the Invention
Malolactic fermentation is a secondary fermentation that occurs in addition to the primary alcoholic fermentation in the production of many wines. The reaction, the decarboxylation of L-malic acid to L-lactic acid, is carried out only by certain species of the genera Lactobacillus, Pediococcus, and Leuconostoc. Malic acid is the major organic acid found in grapes and its decarboxylation to lactic acid results in a significant decrease in the acidity of the wine, which is a matter of some importance in cool grape growing regions where the grape must may be too acidic. Malolactic fermentation also provides bacteriological stability. Once the malolactic fermentation has been completed in wine, no other microbial fermentations occur and this prevents spoilage. The latter is the most important attribute of the malolactic fermentation. A less important attribute is the subtle influence that malolactic fermentation may have on the flavor complexity of the wine.
Malolactic fermentation usually occurs after several months of storage of the wine. This late onset can result in off flavors, and turbidity if it occurs after bottling. The fermentation is slow due to many factors. The initial inoculum is usually small, and more importantly the enviroment of wine is usually not conducive to rapid growth of the malolactic fermenting bacteria. The ethanol and sulfur dioxide concentrations are too high, and the pH, nutrient concentrations and the temperature are too low for optimal growth of these bacteria.
Winemakers often stimulate malolactic fermentation, when it is desirable, through a variety of means. A large inoculum with a lactic acid bacterial strain found to grow with reliability in wine is helpful. Delayed removal of the wine from the yeast sediment, delay in the addition of acidifying agents, low sulfur dioxide concentration, lack of filtration of the wine, and warmer temperatures all encourage malolactic fermentation. These same conditions, however, also encourage spoilage of the wine by other bacteria and yeast species. To prevent such spoilage of wine, it would be desirable to have the malolactic fermentation occur during or shortly after the alcoholic fermentation. The wine could then be adjusted for cellar storage and bottling with no risk of spoilage.
There are a limited number of ways such rapid malolactic fermentation could be induced. Winemakers could add increased dosages of inoculum to induce the fermentation, but such large fermentations would require vast amounts of starter culture. Enzymes isolated from lactic acid bacteria could be used instead of the malolactic fermenting bacteria themselves, but obtaining the enzymes would be difficult and very expensive.
It would thus be desirable to identify and isolate a gene which is able to confer the ability to decarboxylate L-malic acid to L-lactic acid in a suitable host. By introducing this trait to a yeast host capable of fermenting wines, malolactic fermentation could be carried out simultaneously with primary alcoholic fermentation in the production of many wines.
2. Description of the Prior Art
Schutz and Radler (1973) Arch. Mikrobiol. 91:183-202 and Schutz and Radler (1974) Arch. Mikrobiol. 96: 329-339 have purified the malolactic enzyme and report a molecular weight of 150,000 in Lactobacillus plantarum and of 130,000 to 140,000 in Leuconostoc mesenteroides.